PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our laboratory has identified a key role for Galectin-3 (GAL-3 or Lgals3) in driving PASMC proliferation and elevated pulmonary pressures, but the mechanisms underlying GAL-3 upregulation and function remain unknown. Numerous studies have shown that hypoxia inducible factors (HIFs) contribute to PAH in humans and animal models. We have also found increased HIF expression in isolated PA from rat models of PAH and our preliminary data suggests that HIF-2? upregulates GAL-3. GAL-3 contains a BH1 like domain that has an NWGR sequence with high homology to the anti-apoptotic protein, BCL2. Our central hypothesis is that endothelial HIF-2? upregulates GAL-3 which plays an important role in driving PASMC proliferation through repression of apoptosis via the NWGR domain. We will test this hypothesis in 2 specific aims: Aim 1 will examine how HIF-2? upregulates PASMC GAL-3 in PAH. We will administer selective HIF-2? inhibitors in rat models of PAH and expect that GAL-3 expression and hemodynamic indices of PAH will decline. Using a novel GAL-3 floxed mice and a novel lung endothelium- specific AAV that expresses a constitutive form of HIF-2?, we will test the hypothesis that endothelial HIF-2? increases endothelial GAL-3 which drives PASMC proliferation and pulmonary vascular remodeling. We will further investigate a novel mechanism of HIF-2? upregulation via increased expression of the deubiquitinating enzyme Otud7b (Cezanne) in PAH. Aim 2 will determine the importance of the NWGR domain in GAL-3 as a regulator of PASMC apoptosis and proliferation. This will be tested in cultured PASMC from GAL-3 KO rats via re-expression of WT or mutant (G182A) GAL-3 using adenoviral approaches. In vivo, we will generate lung endothelium specific AAVs that express WT or G182A GAL-3 and transduce GAL-3 KO rats prior to administration of MCT. We anticipate that mutant GAL-3 will increase apoptosis and decrease proliferation in PASMC and in vivo this will result in a reduction in pulmonary vascular remodeling and PAH. The proposed project is designed to develop new skills that I do not have significant experience in, primarily animal handling, measurements of hemodynamic variables and experimental design involving animal models. This project will take place at Augusta University within the Vascular Biology Center under the mentorship of Sponsor Dr. David Fulton and Co-Sponsor Dr. Scott Barman. The proposed project is for 3 years of funding and the proposed research would be divided amongst the three years with plans for thesis defense during the final year of funding. Overall we expect this application will highlight the importance of GAL-3 in PAH and will further elucidate the mechanisms underlying increased PASMC proliferation which may be useful in the development of more effective therapeutic approaches.